Breast cancer is responsible for ~43,000 deaths per year, a number which could be reduced dramatically by early detection of the disease. Currently, detection of breast cancer relies primarily on physical examination and conventional mammography. While these procedures have improved early detection of breast cancer and thereby decreased mortality, they still result in relatively high rates of false-positive and false-negative diagnoses. In addition, breast cancer prognosis is based on histological examination and is inadequate for assessing micrometastases. Due to this inability to accurately predict the risk of recurrence, 50% or more of breast cancer patients are unnecessarily treated with adjuvant therapy. A more useful and accurate evaluation of breast cancer could be obtained if these current, observation-based methods were supplemented with a molecular assessment. A number of serum proteins are reported to be altered in individuals with breast cancer when compared with healthy individuals. There is not, however, any known circulating protein that is suitable to define the stage of breast cancer or as a general marker for breast cancer, especially in the early stages of this disease. Mammary ductal cells are the cellular origin for 70% to 80% of breast cancer cases. Nipple aspirate fluid (NAF), which is obtained from non-lactating women, contains proteins secreted directly by these ductal cells. As such, we hypothesize that NAF is a concentrated and selective source of protein biomarkers for breast cancer. Proteomic approaches offer an unbiased way to evaluate NAF as a source of biomarkers and is sufficiently sensitive for small NAF volumes (10 to 50 (l). Therefore, the goals of this proposal are to characterize the proteome of NAF and to undertake preliminary analyses to identify potential protein biomarkers. To accomplish these goals, we propose to undertaken the following specific aims: Aim #1. Characterize the Proteome of Pooled NAFs Using Mass Spectrometry. Aim #2. Identify Potential Protein Biomarkers in Individual NAF Samples from Women with Early-Stage Breast Cancer using Isotopic Labeling and Mass Spectrometry. Upon completion of these aims, we will have provided the first detailed characterization of the NAF proteome and identified a subset of NAF proteins that are potential markers of breast disease. [unreadable] [unreadable]